Live Discussion: Isolation of Single Immunohistochemically Identified Whole Neuronal Cell Bodies from Post-Mortem Human Brain for Simultaneous Anlaysis of Multiple Gene Expression
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Live discussion held 27 February 1998.
Participants: Peter Nelson, Nienwen Chow, Paul Coleman, Kieran Breen, Huntington Potter.
Note: Transcript has been edited for clarity and accuracy.
Nienwen Chow-Paul Coleman: We are here - Chow and Coleman.
Peter Nelson: Hi there. I'm Pete Nelson. I guess most people haven't arrived yet. Maybe we could wait a minute or two before intros and discussion begins.
Nienwen Chow-Paul Coleman: Fine by us. BTW Pete, we just e-mailed our responses to your comments to June K. She suggested we do it this way, so she could append them to your comments. If you wish to discuss any of the points you raised on-line here, let's do so.
Peter Nelson: Maybe in around two minutes we should start, okay?
Nienwen Chow-Paul Coleman: OK. Questions? Points? Answers?
Peter Nelson: Perhaps we should begin with some introductions. My name is Peter Nelson. I'm an MD/PhD finishing my MD at U.Chicago. I'm interested in AD pathobiology.
Kieran Breen: Hi, I'm Kieran Breen, a lecturer from Dundee in Scotland. Peter Nelson: Hi Dr. Breen. I was very interested in the paper. One question that sprung to mind was if you are going to use this technique to look at gene expression in damaged cells, is it possible that only robust cells will survive the initial cell separation.
Nienwen Chow-Paul Coleman: Hi, I'm Nienwen Chow, a Ph.D.. I am working in Paul Coleman's group.
Peter Nelson: I had the same concern. Certainly, there'll be SOME greater frailty in injured cells, by definition.
Nienwen Chow-Paul Coleman: A number of questions have now been raised. With regard to the issue of frailty in sick cells, we are not sure what aspect of the procedure you are referring to. There are other methods of picking cells that are more recently available, such as laser capture micro dissection, that may get around your concerns.
Peter Nelson: But the point is, you're comparing cells of different healthfulness, in re: their RNA. Just by being unhealthy, certain RNAs might be degraded artifactually in a way that has not actual bearing on AD.
Nienwen Chow-Paul Coleman: There also was a private message about asking about more recent, unpublished results. That's OK too.
Peter Nelson: Well? Have you found anything interesting?
Nienwen Chow-Paul Coleman: Let us first address the issue of cells of different health. Is this not an issue with any method that looks at diseased vs. healthy tissue? We'll wait for an answer to this before going on to more recent results.
Peter Nelson: Well, especially one that so directly and dramatically manipulates the cells before analyzing them for something as fragile as RNA.
Kieran Breen: Of course it is, and it is one of the main problems in this research area. However, a technique such as IHC or ISH looks at the cells as they are, while single cell dissection may enrich for a given sub population, thus giving spurious results. Nienwen Chow-Paul Coleman: True, that is why we are exploring other methods of cell isolation. However, we would also emphasize that for selected messages we also confirm results by in situ hybridization.
Peter Nelson: Does it pretty much check out, in relation to ISH? Are there discrepancies?
Kieran Breen: Another question - do you think that it would be possible to differentiate between sick and ordinary cells following dissection? Do they maintain any sort of morphological changes (e.g. do tangle-bearing cells still have tangles?).
Nienwen Chow-Paul Coleman: For example, our aRNA data suggested no change of NF-M expression in AD, contrary to what has been previously demonstrated using Northern blotting. The result of no change in AD was confirmed with in situ, and we now suggest that the earlier studies showing decreased NF-M expression were a consequence of decreased numbers of neurons.
Nienwen Chow-Paul Coleman: Tangled cells do still have tangles after picking. We emphasize that any cell property that can be revealed by IHC or any other procedure, such as Congo red, etc., that leaves the RNA intact can be applied to this technique.
Peter Nelson: Of course, this last point is of considerable interest....
Kieran Breen: This is good, because even if you enrich slightly for more healthy cells, you will, at the end of the day, be choosing the particular cell in which you are interested and you can confirm the cell by its morphology.
Peter Nelson: ...doesn't mean that it's not changed systematically and artifactually, though, Dr. Breen.
Nienwen Chow-Paul Coleman: With regard to more recent results, we do have a manuscript submitted in which we use the aRNA method, and our picking approach to distinguish pyramidal neurons from hippocampus as a function of disease state. Using a multi variate canonical analysis we are able to distinguish brains as to disease state very nicely.
Peter Nelson: Neat! Using the tangle/non-tangle criteria? Or AD vs. PD vs. control, etc.?
Nienwen Chow-Paul Coleman: It was AD vs. control. We put control in parens because although these were clinically control cases ( one lady living at home, paying bills, etc.) on neuropath exam, they were early Braak stages.
Nienwen Chow-Paul Coleman: Hi Hunt.
Huntington Potter: Hi. This is my first chat so I will probably listen for awhile.
Nienwen Chow-Paul Coleman: Please joins as you see fit.
Peter Nelson: Would it be gauche to ask for a preview of how the AD cases differed from controls in their hippocampal neuron RNAs?
Nienwen Chow-Paul Coleman: Long pause while we consider this.
Huntington Potter: One question is whether you are surprised to see ACT in neurons and if you also checked it in astrocytes.
Nienwen Chow-Paul Coleman: First, I would emphasize that the answers one gets depend on the panel of cDNAs used as probes. Given that, we find that the genes that contribute most heavily to the distinction include cyclin D1, an HSP, ACT, wee1.
Kieran Breen: Sorry, have to run. Thanks for the interesting discussion.
Peter Nelson: Cyclin D1 is a very interesting molecule, viz. Esiri's recent work. Peter Nelson: Bye Dr. Breen!
Nienwen Chow-Paul Coleman: Nice to chat with you. Welcome Jhenry.
Peter Nelson: Did you distinguish tangles from non-tangles in your more recent work? Nienwen Chow-Paul Coleman: Not in what we have submitted. This is ongoing now.
Peter Nelson: Is there anything you'd like to say about that later work?
Nienwen Chow-Paul Coleman: We agree that cyclin D1 is interesting, especially in view of the data of Steve Estus indicating its expression induced by APP. The Eseri work also do tie in here. The question is why is cyclin D1 being expressed.
Peter Nelson: What is the variability, from brain to brain, in how good the neurons are for this technique?
Peter Nelson: Oh, in re: the Esiri work, and the reason for the expression of cyclin D1. Perhaps your technique could be used to find the earliest signs of a diseased state, which causes other things to go haywire secondarily.
Nienwen Chow-Paul Coleman: Statistical analysis shows that the % contribution to variability are: 16% due to cell-to-cell, 4.4% brain to brain and 17.6% to disease.
Peter Nelson: ...And all the cells are definitely hippocampal pyramidal neurons of the same Sommer's Sector?
Nienwen Chow-Paul Coleman: Agree with Nelson last comment - that is one of the aims we have. Huntington Potter: I think that I got lost someplace and had to come back in. One further question I have is whether wee 1 is a common mRNA in the brain and whether it is restricted to neurons or glia.
Nienwen Chow-Paul Coleman: We believe that our cell sample included both CA1 and subiculum pyramids.. It is of note that end stage AD brains all clustered very tightly.
Peter Nelson: Dear Mssrs. Coleman, Chow, and Potter: it has been an interesting discussion, but I must go to a class. Thanks very much.
Huntington Potter: By the way. Hi Pete. Yes I remember you and your sheep eps.
Nienwen Chow-Paul Coleman: With regard to Hunt PotterÕs question, we see wee1 in both AD and control neurons. We have not looked at glia yet.
Peter Nelson: Thanks, Hunt!
Nienwen Chow-Paul Coleman: Good to meet you Peter Nelson.
Peter Nelson: Bye! Thanks!
Huntington Potter: Curious. One thing Carmela and I saw some time ago was that ACT antibodies sometimes stained neuronal cell bodies.
Nienwen Chow-Paul Coleman: Well, I guess that our data confirm that.
Huntington Potter: No I am happy good luck with the new exps. over and out.
Nienwen Chow-Paul Coleman: Goodbye.